The ability to investigate structure and dynamics on a micron scale with non-destructive optical probes is key to studies at the single cell level and applications in microfluidics. Confocal microscopy is a technique that provides enhanced resolution due to elimination of out of focus rays by a spatial filter (pinhole) or by multi-photon excitation. For confocal detection, a pinhole is located in the conjugate plane of the focal plane (defined by the collection optics), which enables optical sectioning along the axial direction.
Fluorescence probes employing confocal or other geometries are well established; however they generally require labeling and are limited by photobleaching and quenching. Micro-spectroscopy based on absorption measurements provides a convenient label free way for characterizing an unknown material. Fourier-transform infrared (FTIR) spectroscopic imaging relying on vibrational signatures has numerous applications. Though light scattering has been used recently as a source of contrast in the visible, standard confocal microscopy so far lacks the capability for direct optical absorption profile measurements.
A difficulty for measurements with axial resolution is presented by the ‘missing cone’ problem (see, e.g., M. B. Cannell, A. McMorland and C. Soeller, “Image enhancement by deconvolution”, Handbook of biological confocal microscopy, J. B. Pawley Ed. (Springer, New York, N.Y., 2006), 3rd ed., Chap. 25, pp. 488-500). The optical transfer function is angularly band limited, so that the longitudinal resolution in the axial direction is degraded. To provide spatial discrimination in the axial direction, a confocal laser absorption microscope has been reported. An excitation laser pulse irradiates the sample so that ground-state molecules transit to the excited state, thus creating a spatial distribution of molecules, similarly to what is used in confocal fluorescence. The absorption to higher energy levels is then probed by a monitoring laser beam introduced coaxially. An excited state absorption profile is obtained by scanning the sample. In general the absorption of the laser beam due to electronic transitions from the ground state is assumed to be negligible, although the attenuation of the propagating light could provide a mechanism for contrast in the axial direction.
More simply, the lack of adequate spatial resolution limited the ability to practically measure absorbance in a single cell. Small samples let too much light through the system.
The inventors have recognized the advantages and benefits of a practical and robust solution directed especially to enabling micron-scale axial and lateral resolution absorption spectroscopy to study cells in their native environment and other biological assemblies. For example, the ability to acquire micron-scale absorption measurements of single live erythrocytes in femtoliter volume solutions in micro-capillaries or microchannels, and to determine variations in composition of inhomogeneous samples (e. g. thin films of a few microns), to detect malaria, to monitor blood bank quality by measuring absorption spectrum changes in aging blood cells, to monitor body fluids for pregnancy and AIDS testing, for intrinsic imaging, and other applications and capabilities would be advantageous, especially in microfluidics and nanomaterials characterization. Further advantages and benefits would be obtained with more compact instrumentation.